No abstract

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.

The clpC operon in Staphylococcus aureus comprises four genes, denoted ctsR, mcsA, mcsB and clpC. A mutation within the mcsB gene resulted in hypersensitivity to heavy metal stress, temperature stress, osmotic pressure stress and oxidative stress. This mutation also resulted in sensitivity to variations in pH and lowered expression of the clpC operon under adverse extracellular conditions, as determined by quantitative real-time PCR (qRT-PCR). Additionally, virulence traits such as haemolytic activity, proteolysis, biofilm formation, and evasion from peritoneal fluid killing were substantially reduced in the DmcsB strain. Interestingly, mutated mcsB also caused a significant reduction in expression of virulence determinants hla and saeS. To be a successful pathogen, S. aureus must effectively overcome these types of stresses that are encountered within the host. These data show that an S. aureus strain lacking functional mcsB is stress hypersensitive and therefore less viable when introduced into hostile environments. For the first time, these studies have identified mcsB as a crucial and necessary component of stress and pathogenicity mechanisms.

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.

McsA is a key modulator of stress response in Staphylococcus aureus that contains four CXXC potential metal binding motifs at the N-terminal. S. aureus ctsR operon encodes ctsR, clpC and putative mcsA and mcsB genes. The expression of the ctsR operon in S. aureus was shown to be induced in response to various types of heavy metals such as copper and cadmium. McsA was cloned and overexpressed, and purified product was tested for metal binding activity. The protein bound to Cu(II), Zn(II), Co(II) and Cd(II). No binding with any heavy metal except copper was found when we performed site directed mutagenesis of Cys residues of three CXXC motifs of McsA. These data suggest that two conserved cysteine ligands provided by one CXXC motif are required to bind copper ions. In addition, using a bacterial two-hybrid system, McsA was found to be able to bind to McsB and CtsR of S. aureus and the CXXC motif was needed for the binding. This indicates that the Cys residues in the CXXC motif are involved in metal binding and protein interaction.